K. lactis pyruvate-decarboxylase promoter and use thereof

ABSTRACT

PCT No. PCT/FR93/00694 Sec. 371 Date Feb. 1, 1995 Sec. 102(e) Date Feb. 1, 1995 PCT Filed Jul. 6, 1993 PCT Pub. No. WO94/01569 PCT Pub. Date Jan. 20, 1994The present invention concerns DNA sequences comprising all or part of the K. lactis promoter gene PDC1 or a derivative thereof, and having transcriptional promoter activity. The invention also relates to the use of the sequences for the expression of recombinant genes.

The present invention relates to the field of molecular biology. Moreparticularly, it relates to a novel DNA sequence having atranscriptional promoter activity, expression vectors containing thissequence, and its use for the production of recombinant proteins, and,for example, heterologous proteins. The invention also relates torecombinant cells containing this DNA sequence.

The progress made in the field of molecular biology has enabledmicroorganisms to be modified to make them produce heterologousproteins. In particular, numerous genetic studies have focused on thebacteria E. coli. However, the industrial application of these novelmethods of production is still limited, in particular by the problems ofefficiency of expression of the genes in these recombinantmicroorganisms. In addition, with the aim of increasing the performanceof these production systems, research has been carried out in order toisolate strong promoters enabling high levels of expression ofheterologous proteins to be obtained. For E. coli, the promoters of thetryptophan and lactose operons can be mentioned in particular.

More recently, with the yeast S. cerevisiae, studies have focused onpromoters derived from genes implicated in glycolysis. The studies onthe promoter of the gene of 3-phosphoglycerate kinase PGK (Dobson etal., Nucleic Acid Res. 10, 1982, 2625; Hitzeman et al., Nucleic AcidResearch 1982, 7791), on that of the gene of glyceraldehyde-3-phosphatedehydrogenase GAPDH (Holland et al., J. Biol. Chem. 254, 1979, 9839;Musti et al., Gene 25, 1983, 133), on that of the gene of alcoholdehydrogenase 1 ADH1 (Bennentzen et al., J. Biol. Chem. 257, 1982, 3018;Denis et al., J. Biol. Chem. 25, 1983, 1165), on that of the gene ofenolase 1 ENO1 (Uemura et al., Gene 45, 1986, 65), on that of the geneGAL1/GAL10 (Johnson and Davis, Mol. Cell. Biol. 4 (1984) 1440) or onthat of the gene CYC1 (Guarente and Ptashne, PNAS 78 (1981) 2199) may bementioned especially.

Recently, genetic tools have been developed so as to make use of theyeast Kluyveromyces as host cell for the production of recombinantproteins. The recognition of a two-micron type plasmid native to K.drosophilarum (plasmid PKD1--EP 241 435) has allowed a very efficienthost/vector system for the production of recombinant proteins to beestablished (EP 361 991). However, the promoters used in this systemhave not been optimized until now. In particular, they are essentiallyheterologous promoters, that is to say originating from othermicroorganisms, such as especially S. cerevisiae. This situation canproduce various disadvantages, and especially limit the activity of thepromoter because of the absence of certain elements of thetrans-criptional machinery (for example of trans-activators), exhibit acertain toxicity for the host cell due to an absence of regulation, oraffect the stability of the vector.

Under these conditions, the lack of strong homologous promoters inKluyveromyces constitutes a limiting factor in the industrialexploitation of this expression system.

The Applicant has now identified, cloned and sequenced a region of thegenome of Kluyveromyces lactis presenting a transcriptional promoteractivity (see SEQ ID No. 1 and 2). More precisely, this regioncorresponds to the promoter of the gene encoding the pyruvatedecarboxylase of K. lactis (K1PDC1). This region, or derivatives orfragments of the latter, can be utilized in a very effective manner forthe production of recombinant proteins in the yeasts of the genusKluyveromyces. It is understood that this sequence can also be used inother host organisms.

Moreover, an advantage of the promoter region obtained lies in theabsence of suppression by glucose, allowing use in conventional andindustrial culture media.

One subject of the present invention therefore lies in a DNA sequencecomprising all or part of the sequence SEQ ID No. 1 or of itscomplementary strand, or of a derivative of the latter, and possessing atranscriptional promoter activity.

In the sense of the present invention, derivative is understood asmeaning any sequence obtained from the sequence SEQ ID No. 1 bymodification(s) of genetic and/or chemical nature, retaining a promoteractivity. Modification of genetic and/or chemical nature is understoodas meaning any mutation, deletion, substitution, addition and/ormodification of one or more nucleotides. Such modifications can becarried out with various aims, and especially that of preparing portablepromoters, or that of preparing promoters adapted to expression in aparticular type of vector or host, that of reducing the size, ofincreasing the activity of transcription promoter, of generatinginducible promoters, of improving the level of regulation, or even ofchanging the nature of the regulation. Such modifications can be carriedout, for example, by mutagenesis in vitro, by introduction of additionalcontrol elements or of synthetic sequences, or by deletions orsubstitutions of the original control elements.

When a derivative such as defined above is produced, its transcriptionalpromoter activity can be demonstrated in several ways, and in particularby placing under the control of the sequence studied a reporter genewhose expression is detectable. Any other technique known to the personskilled in the art can quite obviously be used to this effect.

The sequence SEQ ID No. 1 was obtained from a fusion bank betweenfragments of the genome of K. lactis 2359/152 and the lacZ gene of E.coli according to the protocol described in the examples. It isunderstood that the specialist can isolate this region by hybridizationby means of a probe comprising all or part of the sequence SEQ ID No. 1or of its complementary strand. The derivatives according to theinvention can then be prepared from this sequence, as indicated in theexamples.

Another object of the invention relates to a recombinant DNA comprisinga sequence of DNA such as defined above.

This recombinant DNA can contain, for example, the promoter sequence SEQID No. 1 or a derivative of the latter in which is inserted arestriction site facilitating the use of this sequence as a "portable"promoter (SEQ ID No. 4).

Preferentially, this recombinant DNA in addition contains one or morestructural genes. In particular, these can be genes coding for proteinsof pharmaceutical or food-processing interest. By way of example,enzymes (such as, especially, superoxide dismutase, catalase, amylases,lipases, amidases, chymosin, etc.), blood derivatives (such as serumalbumin, alpha- or beta-globin, factor VIII, factor IX, von Willsbrandfactor, fibronectin, alpha-1 antitrypsin, etc.), insulin and itsvariants, lymphokines (such as interleukins, interferons, colonystimulating factors, tumor necrosis is factor (TNF) TGF-B bindingvecector fragment granulocyte colony stimulating factor (G-CSF),granulocyte macrophage colony stimulating factor (GM-CSF), andmacrophage colony stimulating factor (M-CSF), etc.), growth factors(such as growth hormone, erythropoietin, fibroblast growth factor (FGF),epidermal growth factor (EFG), platelet derived growth factor (PDGF),transforming growth factor (TGF), etc.), apolipoproteins, antigenicpolypeptides for the production of vaccines (hepatitis, cytomegalovirus,Epstein-Barr, herpes, etc.), or even fusions of polypeptides such as,especially, fusions comprising an active part fused to a stabilizer part(for example fusions between albumin or fragments of albumin and thereceptor or a part of a virus receptor [CD4, etc.]).

Even more preferentially, the recombinant DNA also contains signalsallowing the secretion of the expression product of the said structuralgene(s). These signals may correspond to natural secretion signals ofthe protein in question, but they may be of a different origin. Inparticular, secretion signals derived from yeast genes can be used, suchas those of the genes of the killer toxin (Stark and Boyd, EMBO J. 5(1986) 1995) or of alpha pheromone (Kurjan and Herskowitz, Cell 30(1982) 933; Brake et al., Yeast 4 (1988) S436).

In a particular embodiment of the invention, the recombinant DNA is partof an expression plasmid which can be of autonomous or integrativereplication.

In particular, autonomous replication vectors can be obtained by usingautonomous replication sequences in the chosen host. Especially, inyeast, they can be replication origins derived from plasmids (pKD1, 2μ,etc.), or even chromosomal sequences (ARS).

The integrative vectors can be obtained especially by using homologoussequences in certain regions of the host genome allowing, by homologousrecombination, integration of the vector.

Another subject of the invention relates to recombinant cells containinga DNA sequence such as defined above.

Advantageously, the cells are chosen from amongst yeasts, and even morepreferentially, amongst yeasts of the genus Kluyveromyces. It isunderstood, however, that the invention covers all the recombinant cellsin which the promoter regions of the invention are active, whether theyare eukaryotic or prokaryotic cells. Thus, among eukaryotic cells,vegetable or animal cells, yeasts or fungi can be mentioned. Inparticular, concerning yeasts, yeasts of the genus Saccharomyces,Pichia, Schwanniomyces or Hansenula can be mentioned. Concerning animalcells, the cells COS, CHO, C127, etc. can be mentioned. Among fungi ableto be used in the present invention, Aspergillus ssp, or Trichodermassp, can be mentioned more particularly. As prokaryotic hosts, bacteriasuch as Escherichia coli can be used, or those belonging to the generaCorynebacterium, Bacillus or Streptoymces.

The transcription promoter activity of the sequences of the invention inthese different hosts can be confirmed, for example, by introducing intothe host cell in question a recombinant DNA comprising, under thecontrol of the promoter sequence studied, a reporter gene whoseexpression can be demonstrated in the host in question.

The recombinant cells of the invention can be obtained by any methodallowing a foreign DNA to he introduced into a cell. It can beespecially transformation, electropotation, conjugation, fusion ofprotoplasts or any other technique known to the person skilled in theart. Concerning transformation, various protocols have been described inthe prior art. In particular, it can be carried out by treating thewhole cells in the presence of lithium acetate and of poly-ethyleneglycol according to the technique described by Ito et al. (J. Bacteriol.153 (1983) 163-168), or in the presence of ethylene glycol and dimethylsulphoxide according to the technique of Durrens et al. (Curr. Genet. 18(1990) 7). An alternative protocol has also been described in the PatentApplication EP 361 991. Concerning electropotation, it can be carriedout according to Becker and Guarentte (in: Methods in Enzymology Vol 194(1991) 182).

Another subject of the invention relates to the use of a sequence suchas defined above for the expression of recombinant genes. The DNAsequences according to the invention can in fact allow production ofrecombinant proteins at high levels.

Advantageously, the sequences of the invention can be used for theexpression of genes encoding proteins of pharmaceutical orfood-processing interest. By way of example, the proteins listed abovemay be mentioned.

The present invention also allows a production process for recombinantproteins to be realized, according to which a recombinant cell such asdefined above is cultured and the protein produced is recovered. By wayof example of protein, the proteins listed above may he mentioned.

Preferentially, the process of the invention is applicable to theproduction of human serum albumin, or one of its molecular variants.Molecular variant of albumin is understood as meaning the naturalvariants resulting from the polymorphism of the albumin, the truncatedforms, or any hybrid protein based on albumin.

Other advantages of the present invention will become apparent fromreading the examples which follow, which may be considered asillustrative and non-limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A & 1B Preparation of the Mini Mu transposon MudIIZK1.

FIG. 2 Restriction map of the Mini Mu transposon MudIIZK1.

FIG. 3 Restriction map of the clone 1D12.

FIG. 4 Restriction map of the 2.05 kb fragment of BamHI-HindIII bearingthe promoter K1PDC1.

GENERAL CLONING TECHNIQUES

The methods conventionally used in molecular biology such as preparativeextractions of plasmid DNA, centrifugation of plasmid DNA in a caesiumchloride gradient, electrophoresis on agarose or acrylamide gels,purification of DNA fragments by electroelution, extraction of proteinswith phenol or with phenol/chloroform, precipitation of DNA in salinemedium with ethanol or isopropanol, transformation in Escherichia colietc. are well known to the specialist and are profusely described in theliterature [Maniatis T. et al., "Molecular Cloning, a LaboratoryManual", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982;Ausubel F. M. et al. (eds), "Current Protocols in Molecular Biology",John Wiley & Sons, New York, 1987].

The restriction enzymes were supplied by New England Biolabs (Biolabs),or Pharmacia and are used according to the recommendations of thesuppliers.

The plasmids of type pBR322 and pUC are of commercial origin (BethesdaResearch Laboratories).

For the ligations, the DNA fragments are separated according to theirsize by electrophoresis in agarose or acrylamide gels, extracted withphenol or with a phenol/chloroform mixture, precipitated with ethanoland then incubated in the presence of the DNA ligase of the phage T4(Boshringer) according to the recommendations of the supplier.

The filling of the prominent 5' ends is carried out by the Klenowfragment of the DNA polymerase I of E. coli (Boshringer) according tothe specifications of the supplier. The destruction of the prominent 3'ends is carried out in the presence of the DNA polymerase of the phageT4 (Biolabs) used according to the recommendations of the manufacturer.The destruction of the prominent 5' ends is carried out by a treatmentmanaged by the nuclease S1.

The directed mutagenesis in vitro by synthetic oligodeoxynucleotides iscarried out according to the method developed by Taylor et al. [NucleicAcids Res. 13 (1985) 8749-8764].

The enzymatic amplification of DNA fragments by the said technique ofPCR [Polymerase-catalysed Chain Reaction, Saiki R. K. et al., Science230 (1985) 1350-1354; Mullis K. B. and Faloona F. A., Meth. Enzym. 155(1987) 335-350] is carried out using a "DNA thermal cycler" (PerkinElmer Cetus) according to the specifications of the manufacturer.

The confirmation of the nucleotide sequences is carried out by themethod developed by Sanger et al. [Proc. Nat1. Acad. Sci. USA, 74 (1977)5463-5467].

The transformations of K. lactis are carried out by any technique knownto the person skilled in the art, and of which an example is given inthe text.

Unless stated otherwise, the bacterial strains used are E. coli DH1(Hanahan D., J. Mol. Biol. 166 (1983) 557) or E. coli JM109::(Mucts)(Daignan-fornier and Bolotin-Fukuhara, Gene 62 (1988) 45).

The strains of yeasts used belong to budding yeasts and moreparticularly to yeasts of the genus Kluyveromyces. The strain K. lactis2359/152 and K. lactis SD6 were particularly used.

The strains of yeasts transformed by the plasmids are cultured inerlenmeyers or in 21 pilot fermenters (SETRIC, France) at 28° C. in richmedium (YPD: 1% yeast extract, 2% Bactopeptone, 2% glucose; or YPL: 1%yeast extract, 2% Bactopeptone, 2% lactose) with constant stirring.

EXAMPLES I--Isolation of the K1PDC1 promoter of K. lactis.

The sequence SEQ ID No. 1 was isolated from a fusion bank betweenfragments of the genome of K. lactis 2359/152 and the lacZ gene of E.coli. This example describes in (A) the preparation of the fusion bank,and in (B) the selection and the characterization of a clone of thisbank bearing the gene promoter of the pyruvate decarboxylase of K.lactis.

A/Preparation of the Fusion Bank

A.1. Preparation of the Mini Mu transposon MudIIZK1 (FIGS. 1 and 2).

The Mini Mu MudIIZK1 was constructed from Mini Mu MudIIZZ1 described byDaignan-Fornier and Bolotin-Fukuhara (Gene 62 (1988) 45). It wasobtained by substituting the replication origin of the mini transposonMudIIZZ1 by a functional replication origin in Kluyveromyces: the originof replication of the plasmid pKD1 (EP231 435).

A.1.1. Construction of a cassette bearing the replication origin of theplasmid pKD1 (fragment s11).

In order to facilitate the subsequent operations, the fragment S11(bearing the replication origin of the plasmid pKD1) was put in the formof a cassette NotI. For this, a derivative of the plasmid pUC18 wasconstructed in which the external sites of the cloning multisite(HindIII and EcoRI sites) were changed to NotI sites. This was done bydigestion with the corresponding enzyme, action of the Klenow enzyme andligation with a synthetic oligonucleotide corresponding to an NotI site[oligo d(AGCGGCCGCT) (SEQ ID No. 5); Biolabs]. The plasmid obtained isdesignated pGM67. The 960 bp fragment S11 obtained by digestion with theenzyme Sau3A of the plasmid KEp6 (Chen et al., Nucl. Acids Res. 114(1986) 4471) was then inserted in the BamHI compatible site of theplasmid pGM67. The plasmid thus obtained designated pGM68 contains, inthe form of a NotI cassette, the fragment S11.

A.1.2. Suppression of the 2μReplication origin of the MudIIZZ1transposon.

The plasmid pGM15 bearing the mini Mu MudIIZZ1 (Daignan-Fornier andBolotin-Fukuhara loc. cit.) was deleted from the 2μregions by digestionby means of the enzyme SalI. The unique SalI site thus obtained was thentransformed to a NotI site by ligation of a synthetic oligonucleotidecorresponding to an NotI site after action of the Klenow enzyme. Theresulting plasmid is called pGM59.

A.1.3. Insertion of the Fragment S11

The cassette NotI bearing the replication origin of the plasmid pKD1(fragment S11) coming from the modified plasmid pUC18 was thenintroduced in the unique NotI site of the plasmid pGM59.

The plasmid obtained, designated pGM83, bears a mini Mu, calledMudIIZK1, which is adapted to the yeast Kluyveromyces lactis, as well asa functional copy of the gene LEU2 of S. cerevisiae capable ofcomplementing a leu2 mutation in K. lactis (K amper et al., Curr. Genet.19 (1991) 109). The restriction map of the mini-mu MudIIZK1 isrepresented in FIG. 2.

A.2. Introduction of the Mini Mu MudIIZK1 into the strain E. colibearing the Mu helper JM109::(Mucts): obtainment of the strainJM109::(Mucts)::(MudIIZK1).

The strain JM109::(Mucts) was transformed by the plasmid pGM83containing the mini mu MudIIZK1 in the presence of calcium chloride.After transformation, transposition was then induced by thermal shockaccording to the technique described by Castilho et al. (J. Bacteriol.158 (1984) 488). The phage lyeate obtained after induction is then usedto superinfect the strain JM109::(Mucts). The strain JM109::(Mucts)being recA, the linear DNA encapsidated by the phage cannot reclose togive a replicative plasmid. The integrants [strainJM109::(Mucts)::(MudIIZK1)] are then selected aschloramphenicol-resistant (Cm^(R)), ampicillin-Sensitive (Amp^(s))clones.

A.3 Preparation of the Genome Bank of K. lactis in E. coli DH1

The high-molecular weight DNA was prepared from the strain K. lactis2359/152, and digested partially by the enzyme Sau3A. The fragments of asize of 4 to 8 kb were recovered on LMP ("Low Melting Point", SEAKEM)agarose gel and cloned in the plasmid pBR322 linearized by BamHI anddephosphorylated by action of calf intestinal phosphatase (Biolabs). 35pools of 1000 colonies in E. coli DH1 were thus produced. The 1000colonies of each pool are ampicillin-resistant andtetracycline-sensitive, which shows that they have all inserted agenomic DNA fragment of K. lactis in pBR322.

A.4. Preparation of the Fusion Bank

A. 4.1 Introduction of the Genome Bank of K. lactis into the strainJM109::(Mucts)::(MudIIZK1).

The plasmid DNA of each pool produced in DH1 is extracted (Maniatis).This DNA is then used to transform the strain JM109::(Mucts)::(MudIIZK1)in the presence of calcium chloride. To be representative of the 1000colonies contained in each pool of the genome bank, more than 3000clones per pool were recovered in the strain JM109::(Mucts)::(MudIIZK1)allowing the transduction.

A.4.2. Transposition of the Mini Mu MudIIZK1

The fusion bank is produced by extensive transposition of the Mini MuMudIIZK1 in the plasmids forming the genomic DNA bank of K. lactis. Themini-muductions were carried out according to the protocol described byCastilho et al. (J. Bacteriol. 158 (1984) 488) and the transductantswere selected on LBAC selective medium (LB medium (Gibco BRL)supplemented with 50 mg/l of ampicillin and 30 mg/l of chloramphenicol),the marker Amp^(R) being contributed by the plasmid and the markerCm^(R) by the mini-mu. For each pool, transpositions are done in series,and between 10,000 and 20,000 transductants are recovered per pool. TheDNA of the transductants is then extracted from a preparation of 100 ml,purified by precipitation with polyethylene glycol (Maniatis et al.,1989) and resuspended in 100 μl of water. This DNA was then used totransform K. lactis and select clones bearing promoters.

B/Isolation of the K1PDC1 Promoter of K. lactis

The fusion DNA prepared above was utilized to transform, byelectroporation, a receptor strain of K. lactis. This receptor strain,designated SD6, bears the mutations leu2 (corresponding to the selectionmarker of the mini-mu MudIIZK1) and lac4-8. This last mutation preventsthe strain from growing on a medium containing lactose as the onlysource of carbon, but it can be complemented by the superexpression ofthe lacZ gene of E. coli encoding β-galactosidase (Chen et al., J. BasicMicrobiol. 28 (1988) 211). Therefore the expression of a protein fusedto β-galactosidase may allow the growth of the strain SD6 on lactoseafter transformation. This positive screen was used to select rapidlyclones bearing strong promoters.

B.1. Construction of the Receptor Strain K. lactis SD6.

The strain SD6 (Chen et al., Mol. Gen. Genet. 233 (1992) 97) wasobtained by growth of the strain K. lactis CXJ1-7A (a, lac4-8, ura3A,ade1--1, K1, K2, pKD1) (Chen and Fukuhara, Gene 69 (1988) 181) with thestrain AWJ-137 (leu2, trp1, homothallic) (K amper et al., Curr. Genet.19 (1991) 109), and selection of the spores having the genotype ADE⁺,uraA, leu2, lac4-8. As the spores obtained were not capable ofregenerating after transformation by protoplasts, a backcrossing wasdone with the strain CXJ1-7A. After sporulation en masse, the spores ofthe selected genotype were tested by transformation in lithium chloridewith the plasmid KEp6 according to a technique derived from thatdescribed by Ito et al. (J. Bacteriol. 153 (1983) 163) (theconcentration of LiCl is 20 mM, being 10 times less than that used byIto for S. cerevisiae). The strain CXJ1-7A was used as transformationcontrol.

The strain SD6, selected on these criteria, transforms correctly: 1 to3×104 transformants per μg of DNA; and the transformants have asatisfactory stability: 30 to 40% of the colonies retain the [Ura⁺ ]phenotype after 6 generations in non-selective medium.

B.2. Isolation of the K1PDC1 promoter.

The strain SD6 was transformed by electropotation according to Beckerand Guarante (in Methods in Enzymology vol 194 (1991) 182) (Jouanapparatus; 2500 V/cm; 80-100 ng of DNA/transformation) with the DNA of11 pools of transductants obtained in A.4.2. (corresponding to a bank of11,000 clones in E. coli). After regenerating for 5 hours in YPD medium(yeast extract 10 g/l; peptone 10 g/l; glucose 20 g/l), the cells werespread on minimum lactose medium. The transformants capable of growingon lactose were restreaked and, tot each clone, the plasmid wasextracted, amplified in E. coli, and, after rapid verification of therestriction map of the vector and of the mini-mu, used to retransformthe yeast SD6. Among the clones of K. lactis obtained afterretransformation, one of them, the clone 1D12, was studied byrestriction (see FIG. 3) and by analysis of the sequence of the Junctionbetween the protein of K. lactis and β-galactosidase. For this, thesequence of the junction starting from the lacZ end of the mini-mu(double-stranded sequence) was determined by sequencing by means of thefollowing oligonucleotide situated at -59 nucleotides from the Junction:5'-CTGTTTCATTTGAAGCGCG-3'(SEQ ID No. 3)

The analysis of the protein sequence deduced from the nucleotidesequence thus obtained by comparison with the sequences of protein banksof other yeasts or eukaryotes (Ganbank, MIPS, EMBL, etc.), shows thatthe sequence borne by the clone 1D12 corresponds to the promoter of thepyruvate decarboxylase gene of K. lactis. The BamHI-HindIII fragment of2.05 kb containing the region upstream of the fusion was then subclonedinto the vector Bluescript KS+(Stratagene), a restriction map was done(FIG. 4), and the sequence was determined by sequential deletions on 1.2kb (SEQ ID No. 1). The obtainment of sequence elements also allows thespecialist to prepare specific probes and to reclone the promoter regionof the invention by hybridization according to the conventionaltechniques of molecular biology.

II--Transformation of Kluyveromyces

Various techniques permitting the introduction of DNA into the yeast canbe used.

Advantageously, the different strains of Kluyveromyces used weretransformed by treating the whole cells in the presence of lithiumacetate and of polyethylene glycol according to the technique describedby Ito et al. (J. Bacteriol. 153 (1983) 163-168). The transformationtechnique described by Durrens et al. (Curr. Genet. 18 (1990) 7) usingethylene glycol and dimethyl sulphoxide was likewise used. It is alsopossible to transform yeasts by electroporation, for example accordingto the method described by Karube et al. (FEBS Letters 182 (1985) 90).

An alternative protocol has already been described in detail in theapplication EP 361 991.

III--Use of the Promoter SEQ ID No. 1 for Expression of HeterologousGenes.

The transcriptional promoter activity of the region of K. lactisdescribed in SEQ ID No. 1 was recognized even at the time of itsisolation by its capacity to induce the complementation of the lac4-8mutation of the strain SD6. This capacity in fact results from theexpression of the lacZ gene of E. coli, and demonstrates by the sametoken the capacity of expression of heterologous genes.

IV--Construction of a Portable K1PDC1 Promoter (SEQ ID No. 4)

A portable promoter is prepared by PCR, by insertion in the 2.05 kbBamWI-HindIII fragment of a HindIII restriction site in the +1 positionwith respect to the codon ATG of the gene K1PCD1 and of the MluI andSalI restriction sites at 1165 bp upstream (SEQ ID No. 4). The PCRproduct is cloned in the vector pCRII (Invitrogen) to generate theplasmid pYG175, allowing the promoter to be released by simpleMluI-HindIII digestion, thus facilitating the cloning in an expressionvector.

An expression vector of human serum albumin is then prepared from theplasmid pYG1018 as follows: the plasmid pYG1018 contains theprepro-albumin gene under the control of the LAC4 promoter. It derivesfrom the vector pYG1023 described in the Patent Application EP 402 212by deletion of the BssHII-MluI fragment bearing the K1PGK gene. 5 μg ofthe pCRII Promoter and pYG1018 vectors are digested with 60 units ofHindIII and of MluI. After migration on agarose gel at 0.8%, the bandcorresponding to the promoter PDC1 (approximately 1.2 kb), the bandcorresponding to the vector part (approximately 9 kb) and the bandcorresponding to the cDNA of the albumin (approximately 2 kb) areelectroeluted. Ligation to 3 partners (following the buffer andtemperature recommendations defined by the supplier New England Biolabs)is then carried out with 1 μl of promoter DNA, 1 μl of vector DNA and 2μl of albumin DNA. After transformation in E. coli (Chung et al. NAR 16(1988) 3580), the plasmid DNA of the transformants is prepared accordingto the technique of alkaline lysis on SDS of Birboim and Doly (NAR 6(1979) 1513) modified by Ish-Horowicz and Burke (NAR 9 (1981) 2989).After enzymatic digestion, the plasmid possessing the good restrictionprofile is isolated. This plasmid is designated pYG181.

The strain K. lactis CBS 293.91 was transformed by pYG181 under theconditions described in Example II. The production of albumin by severaltransformants is tested according to the technique described in EP 361991. The quantity of albumin secreted by the transformants is similar(50-100 mg/l).

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 5                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1239 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Kluyveromyces lactis                                            (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1177..1239                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AAGCTTCCAGTCACATGACCTAGAATGCATATATTGTCCCGGGTAATATATAGCAACCGT60                CTTCTCTCTGCTTATCTCTGGTGCAGCCTCCTAGTTTTTCCGAAAAGTTTCTTTTTCTCC120               AAAATTCTCCGAAAATCGTCAGATATCCAGATATGCAACATAGAGGGAAGATACACCGTT180               ATATCGATTTCAAAATGCAAATAACGGCAACTAAGCCCATCACTCTCAGCACGTTTCTTT240               TCCATGCACGTCGTTCCCACTGATGATTCCTCGGATCAGTTGATGCGATTTTTAACAACA300               GCCAAAGCAGACATCCTCCAAGCCGTATCCCAGACAAATACAACTCTATCTCTGTGAGGG360               ATTCGTGTAAACTGGAATTCAGCTGAGGTATTTCTTTTTACAGGATTGCGCTTCCAGTAA420               ATTCGGTCTGTTCCATTGCGACTTGTTGAATGGCATTGGCGATGCCAATCGCAATTCCAT480               GGGCAACGAGTAAGTAAGTCTCTTCTACATCGTCCTATTCCTTTCCTAATGTGAGCTCTT540               TTTACCTTTTGGTACCCATACAACGAGTGTCATGGGGAGAGGAAGAGGAAGATTTTAGAT600               GTAGTGAAAGTGAATAGATGAAATGGGAAATGGGCGTGTGGTGCGGTTTCTGTGGTATAG660               AAAATCGGAAATGAGAGTCAACCAAGGTAAAAGGTGGGATAATTTCATGGTATTGGAAAT720               TATTCAAATTAGCTAACCATTGCTGGCCCTTGTGCGTGTCCTGTATTTTGCATATTCTGT780               ATTTGCTATGTAGAACCATGGGATAGATTATGCAATATTTGTGAAAAGATTCAACCTTTT840               AAGGTGTATACGTAGTATTGGCCCATGAATATCTTAAGGTTGGAATAGTAGTACCACTTG900               TGTCAATTATCCATTAAGGGGGGAGAGTGAGGTGAGAGGCAGGGCTGGCTGGGTGGTGAA960               TATGACCTTTCTATTTTTTTTAGTTAACTCAGATAAAGTATAAATACATGGGCATGATTA1020              TCTGTAATGGCTAGAGTTTCCCATCATGTCTTAATCATAATCTTAATTATATACTTTTGA1080              TTACCCTCAAAAACCATCCACTAAAGCCAAACATATTATAGTATTAACTATTAATATTAA1140              GGATAAAACTACAACTCAAAACCAACTTAAATTACAATGTCTGAAATTACATTA1194                    MetSerGluIleThrLeu                                                            15                                                                            GGTCGTTACTTGTTCGAAAGATTAAAGCAAGTCGAAGTTCAAACC1239                             GlyArgTyrLeuPheGluArgLeuLysGlnValGluValGlnThr                                 101520                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetSerGluIleThrLeuGlyArgTyrLeuPheGluArgLeuLysGln                              151015                                                                        ValGluValGlnThr                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CTGTTTCATTTGAAGCGCG19                                                         (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1184 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Kluyveromyces lactis                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ACGCGTGTCGACAGCTTCCAGTCACATGACCTAGAATGCATATATTGTCCCGGGTAATAT60                ATAGCAACCGTCTTCTCTCTGCTTATCTCTGGTGCAGCCTCCTAGTTTTTCCGAAAAGTT120               TCTTTTTCTCCAAAATTCTCCGAAAATCGTCAGATATCCAGATATGCAACATAGAGGGAA180               GATACACCGTTATATCGATTTCAAAATGCAAATAACGGCAACTAAGCCCATCACTCTCAG240               CACGTTTCTTTTCCATGCACGTCGTTCCCACTGATGATTCCTCGGATCAGTTGATGCGAT300               TTTTAACAACAGCCAAAGCAGACATCCTCCAAGCCGTATCCCAGACAAATACAACTCTAT360               CTCTGTGAGGGATTCGTGTAAACTGGAATTCAGCTGAGGTATTTCTTTTTACAGGATTGC420               GCTTCCAGTAAATTCGGTCTGTTCCATTGCGACTTGTTGAATGGCATTGGCGATGCCAAT480               CGCAATTCCATGGGCAACGAGTAAGTAAGTCTCTTCTACATCGTCCTATTCCTTTCCTAA540               TGTGAGCTCTTTTTACCTTTTGGTACCCATACAACGAGTGTCATGGGGAGAGGAAGAGGA600               AGATTTTAGATGTAGTGAAAGTGAATAGATGAAATGGGAAATGGGCGTGTGGTGCGGTTT660               CTGTGGTATAGAAAATCGGAAATGAGAGTCAACCAAGGTAAAAGGTGGGATAATTTCATG720               GTATTGGAAATTATTCAAATTAGCTAACCATTGCTGGCCCTTGTGCGTGTCCTGTATTTT780               GCATATTCTGTATTTGCTATGTAGAACCATGGGATAGATTATGCAATATTTGTGAAAAGA840               TTCAACCTTTTAAGGTGTATACGTAGTATTGGCCCATGAATATCTTAAGGTTGGAATAGT900               AGTACCACTTGTGTCAATTATCCATTAAGGGGGGAGAGTGAGGTGAGAGGCAGGGCTGGC960               TGGGTGGTGAATATGACCTTTCTATTTTTTTTAGTTAACTCAGATAAAGTATAAATACAT1020              GGGCATGATTATCTGTAATGGCTAGAGTTTCCCATCATGTCTTAATCATAATCTTAATTA1080              TATACTTTTGATTACCCTCAAAAACCATCCACTAAAGCCAAACATATTATAGTATTAACT1140              ATTAATATTAAGGATAAAACTACAACTCAAAACCAACTAAGCTT1184                              (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AGCGGCCGCT10                                                                  __________________________________________________________________________

We claim:
 1. A DNA sequence selected from the group consisting of:(a)the sequence presented in SEQ ID NO: 1 or SEQ ID NO 4; (b) acomplementary strand of the sequence presented in SEQ ID NO: 1 or SEQ IDNO 4; and (c) a fragment of the sequence of (a) or (b); wherein said DNAsequence possesses transcriptional promoter activity.
 2. A recombinantDNA comprising a DNA sequence according to claim
 1. 3. A recombinant DNAaccording to claim 2, further comprising one or more structural genes.4. A recombinant DNA according to claim 3, further comprising signalsenabling secretion of expression products of said one or more structuralgenes.
 5. A recombinant DNA according to claim 3, wherein the one ormore structural genes encode proteins of pharmaceutical orfood-processing interest.
 6. A recombinant DNA according to claim 2,wherein said recombinant DNA is an autonomous or integrative replicationvector.
 7. A recombinant cell containing a DNA sequence according toclaim
 1. 8. A recombinant cell according to claim 7, characterized inthat said cell is a yeast.
 9. A recombinant cell according to claim 8,characterized in that said cell is a yeast of the genus Kluyveromyces.10. A process for the production of recombinant proteins, comprisingculturing a recombinant cell according to claim 7 and recovering theproteins produced.
 11. A process according to claim 10, wherein saidproteins are of pharmaceutical or food-processing interest.
 12. Aprocess according to claim 10, wherein the protein is human serumalbumin or a natural variant of human servin albumin resulting frompolymorphism.